To study intrinsic SR Ca2+ cycling in SANC and VM without interference of ionic channels and to test our hypothesis we employed saponin-permeabilized rabbit SANC and VM. (1) At similar physiological intracellular Ca2+ concentrations local Ca2+ releases were large and rhythmic in permeabilized SANC, but were small and random in permeabilized VM. SANC spontaneously released more Ca2+ from the sarcoplasmic reticulum than did VM, despite comparable sarcoplasmic reticulum Ca2+ content in both cell types. This ability of SANC to generate more robust and rhythmic local Ca2+ releases was associated with increased abundance of sarcoplasmic reticulum Ca2+-ATPase (SERCA), reduced abundance of the SERCA inhibitor phospholamban (PLB), and increased Ca2+-regulated PKA- and CaMKII-dependent phosphorylation of PLB and RyR. The increased phosphorylation of RyR in SANC may facilitate Ca2+ release from the sarcoplasmic reticulum, whereas Ca2+-dependent increase in phosphorylation of PLB relieves its inhibition of SERCA, augmenting the pumping rate of Ca2+ required to support robust, rhythmic local Ca2+ releases. The differences in Ca2+ cycling between SANC and VM provide insights into the regulation of Ca2+ clock-like intracellular Ca2+-cycling that drives normal automaticity of cardiac pacemaker cells. (2) To test our second idea we elevated phosphorylation of sarcoplasmic reticulum -associated proteins, PLB and RyR and studied spontaneous Ca2+ release characteristics in permeabilized rabbit VM at physiological intracellular Ca2+ concentrations, prior to and following inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA. An increase in phosphorylation level of Ca2+-cycling proteins converted stochastic Ca2+ sparks into robust, periodic Ca2+ releases similar to ones observed in SANC. Thus, a Ca2+ clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca2+ cycling increases and spontaneous local Ca2+ release becomes partially synchronized. (3) Finally, we verified role of basal PKA- and CaMKII-dependent protein phosphorylation for spontaneous beating of intact rabbit SANC. Cardiac pacemaker cells had a high basal level of both PKA- and CaMKII-dependent protein phosphorylation, which was replicated in basal phosphorylation of Ca2+ cycling proteins PLB and RyR. Specifically, basal level of PLB phosphorylation at PKA-dependent Ser16 site was substantially higher in SANC than in VM; phosphorylation of PLB at CaMKII-dependent Thr17 site was also markedly elevated in SANC compared to VM, and ratio of P-PLB/total PLB exceeded that in VM by 3-fold. Basal phosphorylation of RyR at CaMKII-dependent Ser2815 site in the rabbit SA node was markedly higher than that in ventricular tissue, and ratio of P-Ser2815/total RyR in the SA node surpassed that in the ventricle by 10-fold. Insights from these studies may help in the design of gene- or cell-based biological pacemakers that could be used instead of electronic devices in individuals with sick sinus syndrom which is primarily a disease of the seniors and increases in an exponential manner with aging.